Method and apparatus for making fibers for sectioned arrays

ABSTRACT

A method and apparatus for making a fiber, especially a fiber adapted for use in a sectioned array, are provided according to the invention. The method includes a step of supplying a composition into a mold or tubing wherein the composition solidifies in the mold or tubing. The method further includes a step of allowing the composition to solidify and form the fiber. The method further includes a step of placing a predetermined elongation force onto an end of the fiber, the predetermined elongation force causing an elongation and reduction in cross-section of the fiber and causing a separation of the fiber from an interior surface of the mold or tubing. The method further includes a step of substantially maintaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing.

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to a method and apparatusfor making fibers for sectioned arrays.

[0002] Analysis of the components of a particular substance or compoundmay be an important and difficult process. The substance may have manycomponents, and the components may be present in widely varyingquantities. There is a constant need for the ability to quickly, easily,and accurately determine the components of a test sample. One area forsuch analysis is medical diagnostics, where a patient sample may betested for diseases, toxins, drug levels, hormone levels, etc. Anotherarea in which component analysis is needed is, for example, detection ofproteins, nucleic acids, etc. Additional uses include testing andanalysis of any organic or inorganic compound.

[0003] Available test arrays reflect the ongoing need for small devicesor test kits that test for a wide array of molecules or macromolecules.The need is for simple, high output testing, including the capabilityfor automated testing and detection in a quick and reliable fashion.

[0004] The prior art has attempted to meet this need by creating arraysor microarrays that contain large groupings of binding agents of varyingtypes. When exposed to a test sample, molecules or macromolecules ofinterest may bind to these various binding agents. The binding agentsmay then be detected in various ways, such as illumination by light anddetection of light emitted in response (the binding agents may includedyes).

[0005] Microarrays are known in the art and are commercially availablefrom a number of sources. Microarrays have been used for a number ofanalytical purposes, typically in the biological sciences. An array isessentially a two dimensional sheet where different portions or cells ofthe sheet have different biomolecule elements, such as, nucleic acids orpeptides, bound thereto. Microarrays are similar in principle to othersolid phase arrays except that assays involving such microarrays areperformed on a smaller scale, allowing many assays to be performed inparallel.

[0006] The prior art methodology for preparation of protein chipsrequires preparation, use and reuse of large numbers of proteins insolution. Proteins in solution are unstable and deteriorate over time.Even if frozen, repeated use may involve repeated freeze-thaw cyclesthat denature certain proteins. By contrast, immobilized proteins havebeen shown to be stable over long periods of time.

[0007] In the prior art, two-dimensional arrays of macromolecules aremade either by depositing small aliquots on flat surfaces underconditions which allow the macromolecules to bind or be bound to thesurface, or the macromolecules may be synthesized on the surface usinglight-activated or other reactions. Prior art array formation methodsinclude using printing techniques to produce such arrays. Some methodsfor producing arrays have been described in “Gene-ExpressionMicro-Arrays: A New Tool for Genomics”, Shalon, D., in FunctionalGenomics: Drug Discovery from Gene to Screen, IBC Library Series,Gilbert, S. R. & Savage, L. M., eds., International BusinessCommunications, Inc., Southboro, Mass., 1997, pp 2.3.1.-2.3.8; “DNAProbe Arrays: Accessing Genetic Diversity, Lipshutz, R. J., inFunctional Genomics: Drug Discovery from Gene to Screen, IBC LibrarySeries, Gilbert, S. R. & Savage, L. M., eds., International BusinessCommunications, Inc., Southboro, Mass., 1997, pp 2.4.1-2.4.16;“Applications of High-Throughput Cloning of Secreted Proteins andHigh-Density Oligonucleotide Arrays to Functional Genomics:,Langer-Safer, P. R., in Functional Genomics: Drug Discovery from Gene toScreen, IBC Library Series, Gilbert, S. R. & Savage, L. M.,International Business Communications, Inc., Southboro, Mass., 1997, pp2.5.13; Jordan, B. R., “Large-scale expression measurement byhybridization methods: from high-densities to ‘DNA chips’”, J. Biochem.(Tokyo) 124: 251-8, 1998; Hacia, J. G., Brody, L. C. & Collins, F. S.,“Applications of DNA chips for genomic analysis”, Mol. Psychiatry 3:483-92, 1998; and Southern, E. M., “DNA Chips: Analyzing sequence byhybridization to oligonucleotides on a large scale”, Trends in Genetics12: 10-5, 1996.

[0008] The terms “arrays” and “microarrays” are used somewhatinterchangeably differing only in their general size. The presentinvention involves the same methods for making and using either. Eacharray typically contains many cells (typically 100-1,000,000+) whereineach cell is at a known location and contains a specific component ofinterest. Each array therefore contains numerous different components ofinterest.

[0009] The number of different cells and therefore the number ofdifferent biochemical molecules being tested simultaneously on one ormore microarrays can range into the thousands. Commercial microarrayplate readers typically measure fluorescence in each cell and canprovide data on thousands of reactions simultaneously, thereby savingtime and labor. A representative example of the dozens of patents inthis field is U.S. Pat. No. 5,545,531.

[0010] Many biochemical analyses require that the analytical procedurehave wide dynamic range. Enzyme and immunochemical assays are often doneby determining the course of a reaction over a period of time, or bydoing the analyses on a series of dilutions. In addition, parallelanalyses using standards and blanks (controls) are required and areuniversally included. Large numbers of standardized inexpensive biochipswill be required to meet these needs. These biochips may incorporatereactants of different classes that can, for example, detect and measureantigens, drugs, nucleic acids or other analytes simultaneously.

[0011] Regardless of the technique, each prior art microarray isindividually and separately made, typically is used only once and cannotbe individually precalibrated and evaluated in advance. Hence, the priorart depends on the reproducibility of the production system to produceerror-free arrays. These factors have contributed to the high cost ofcurrently produced biochips or microarrays, and have discouragedapplication of this technology to routine clinical use.

[0012] One of the advantages of microarrays is that they may bemachine-read and the result may be digitally stored and/or processed.For reading or scanning microarrays, charged coupled device (CCD)cameras are widely used. However, in one proposed variation, an array islocated at the end of a bundle of optical fibers with the nucleic acidor antibody/antigen attached to the end of the optical fiber. Detectionof fluorescence may then be performed through the optical fiber.

[0013] Scanning or reading arrays are routinely produced in which glassor plastic fibers are arrayed in parallel in such a manner that allremain parallel, and an optical image may be transmitted through thearray. Parallel arrays may also be made of hollow glass fibers, and thearray sectioned normal to the axis of the fibers to produce channelplates used to amplify optical images. A microarray may be brought intoproximity with a camera or scanner, and an image or images may becaptured.

[0014] Arrays may have an entire set of antigens/antibodies, etc., inthe various cells, along with controls to effectively screen bloodsamples for common bloodborne diseases before donated blood is providedfor transfusion. Likewise, certain symptoms have a number of commoncauses that may be simultaneously screened for using arrays. Forexample, urinary tract infections are common and may be caused by alarge number of different bacteria of varying sensitivity to variousantibiotics. The simultaneous testing for numerous factors would saveconsiderable time and expense.

[0015] In the prior art, biochemical molecules on microarrays have beensynthesized directly at or on a particular cell on the microarray.Alternatively, preformed molecules have been attached to particularcells of the microarray by chemical coupling, adsorption or other means.

[0016] Regulatory approval of clinical tests and systems and methods formaking them is required. When chips are fabricated usingphotolithography and other technology derived from electronic chipmaking, as has been done in the prior art, the cost of formingindividual chips is extraordinarily high. In addition, the possibilityof error when chips are individually made is very high. Since chips areindividually made and used only once, quality control is difficult andthere is no good way of proving that any given chip is satisfactory. Thebest that can be done is to test a large fraction of a batch at random.

[0017] In a recent development, fibers containing test materials areformed and used to make sectioned arrays. Immobilized enzymes have beenprepared in fiber form from an emulsion as disclosed in Italy Pat. No.836,462. Antibodies and antigens have been incorporated into solid phasefibers as disclosed in U.S. Pat. No. 4,031,201. A large number of otherdifferent immobilization techniques have been used and are well known inthe fields of solid phase immunoassays, nucleic acid hybridizationassays and immobilized enzymes; see, for example, Hermanson, Greg, T.Bioconjugate Techniques. Academic Press, N.Y. 1995, 785 pp; Hermanson,G. T., Mallia, A. K. & Smith, P. K. Immobilized Affinity LigandTechniques. Academic Press, N.Y., 1992, 454 pp; and Avidin-BiotinChemistry: A Handbook. D. Savage, G. Mattson, S. Desai, G. Nielander, S.Morgansen & E. Conklin, Pierce Chemical Company, Rockford Ill., 1992,467 pp.

[0018] In the prior art, a fiber is typically formed of a polymerizablematerial injected or inserted into a plastic tubing to form an encasedfiber. In order to obtain a relatively dense array of a small physicalsize, a tubing may have an inner diameter as small as about 200 microns.

[0019] In the prior art, the fiber is not removed from the tubing. Thishas several drawbacks.

[0020] Due to the inclusion of the tubing and the fiber in the sectionedarray, the size of the sectioned array may be larger than desired.Alternatively, the number of test sites may have to be reduced. Inaddition, the presence of the tubing surrounding the fiber may reducecontact between the binding agents and the fiber. Likewise, thesectioned array also suffers from a reduced surface area with which tointeract with a test sample.

[0021] What is needed therefore are improvements in making fibers forsectioned arrays.

SUMMARY OF THE INVENTION

[0022] A first method for making a fiber, especially but not necessarilya fiber adapted for use in a sectioned array, is provided according tothe invention. The method comprises a step of supplying a compositioninto a mold, the mold being of any desired shape, e.g., having across-section which is round, square or oval, and can be tubing. Thecomposition is one which will solidify in the mold. The composition maybe a polymerizable composition which polymerizes within the mold or itmay be a composition which is liquid and then solidifies upon cooling,e.g., a wax. The composition may also be a polymer which is not yetsolid but becomes a solid in the presence of a complexing agent such asa calcium, phosphate or other ion which can be diffused into the polymersolution within the mold or tubing. Examples of such polymers arealginates and other gels and gums. The method further comprises a stepof allowing the composition to solidify (this term encompassing theformation of a solid via a polymerization step, a cooling step, acomplexing step, etc.) and form the fiber. The method further comprisesa step of placing a predetermined elongation force onto an end of thefiber, the predetermined elongation force causing an elongation andreduction in diameter of the fiber and causing a separation of the fiberfrom an interior surface of the mold or tubing. The method furthercomprises a step of substantially maintaining the predeterminedelongation force to propagate the separation through the mold or tubinguntil the fiber is completely separated from the interior surface of themold or tubing. The method may optionally include a step of slitting themold or tubing.

[0023] A second method for making a fiber adapted for use in a sectionedarray is provided according to the invention. The method comprises astep of supplying a composition into a mold or tubing, wherein thecomposition is one which will solidify in the mold or tubing. The methodfurther comprises a step of allowing the composition to solidify andform the fiber. The method further comprises a step of exposing an endof the fiber. The method further comprises a step of placing apredetermined elongation force onto the end of the fiber, thepredetermined elongation force causing an elongation and reduction indiameter of the fiber and causing a separation of the fiber from aninterior surface of the mold or tubing. The method further comprises anoptional step of slitting the mold or tubing. The method furthercomprises a step of substantially maintaining the predeterminedelongation force to propagate the separation through the mold or tubinguntil the fiber is completely separated from the interior surface of themold or tubing.

[0024] A third method for making a fiber adapted for use in a sectionedarray is provided according to the invention. The method comprises astep of supplying a composition which can solidify into a mold or tubingusing a plunger apparatus communicating with the mold or tubing. Themethod further comprises a step of allowing the composition to solidifyand form the fiber in the mold or tubing and to solidify in the plungerapparatus. The method further comprises a step of placing apredetermined elongation force onto an end of the fiber using theplunger apparatus, the predetermined elongation force causing anelongation and reduction in diameter of the fiber and causing aseparation of the fiber from an interior surface of the mold or tubing.The method further comprises a step of substantially maintaining thepredetermined elongation force to propagate the separation through themold or tubing until the fiber is completely separated from the interiorsurface of the mold or tubing. The method may optionally include a stepof slitting the mold or tubing.

[0025] A fourth method for making a fiber and a sectioned array isprovided according to the invention. The method comprises the steps ofsupplying a composition which can solidify into a mold or tubing;allowing the composition to solidify and form the fiber, and placing apredetermined elongation force onto an end of the fiber. Thepredetermined elongation force causes an elongation and reduction indiameter of the fiber and causes a separation of the fiber from aninterior surface of the mold or tubing. The method further comprises astep of substantially maintaining the predetermined elongation force topropagate the separation through the mold or tubing until the fiber iscompletely separated from the interior surface of the mold or tubing.The method further comprises the steps of substantially aligning aplurality of fibers into a bundle, affixing fibers of the bundle,sectioning the bundle, and affixing a section piece to a substrate toform the sectioned array.

[0026] An apparatus for making a fiber adapted for use in a sectionedarray is provided according to the invention. The apparatus comprises aknife block having a passage therethrough of a size to accommodate atubing having a fiber therein. The apparatus further comprises at leastone knife adjustably held in the knife block and positionable so thatthe at least one knife slits the tubing when the tubing is fed throughthe knife block. The apparatus further comprises a tensioning devicecapable of gripping the fiber and placing a predetermined elongationforce on the fiber. The predetermined elongation force propagates anelongation and reduction in diameter of the fiber in the tubing andcauses a separation of the fiber from an interior surface of the tubing.The predetermined elongation force also pulls the tubing and the fiberthrough the knife block, where the tubing is slit.

[0027] The above and other features and advantages of the presentinvention will be further understood from the following description ofthe preferred embodiment thereof, taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 shows a tubing containing a fiber formed therein;

[0029]FIG. 2 shows a cut-away portion of the tubing that has been cutaway to expose the end of the fiber;

[0030]FIG. 3 shows the fiber removal according to the present invention;

[0031]FIG. 4 shows a plunger apparatus such as a commonly availablesyringe;

[0032]FIG. 5 shows an apparatus for forming an essentially continuousfiber;

[0033]FIG. 6 shows detail of one knife block half;

[0034]FIG. 7 shows detail of one knife block half with one or moreknives in place; and

[0035]FIG. 8 shows a cut depth adjuster.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] The terms “binding component”, “molecule of interest”, “agent ofinterest”, “ligand” or “receptor” may be any of a large number ofdifferent molecules, biological cells or aggregates, and the terms areused interchangeably. Each binding component is immobilized at a cell,site, or element of the array, and binds to an analyte being detected.Therefore, the location of an element or cell containing a particularbinding component determines what analyte will be bound. Proteins,polypeptides, peptides, nucleic acids (oligonucleotides andpolynucleotides), antibodies, ligands, polysaccharides, microorganisms,receptors, antibiotics, test compounds (particularly those produced bycombinatorial chemistry), bacteria, viruses, or plant and animal cellsand organelles or fractions of each may each be a binding component ifimmobilized in an element of a microarray. Each of the substances abovemay also be considered as analytes if they bind to a binding componenton a chip.

[0037] When a molecule of interest has a high molecular weight, it isreferred to as a “macromolecule”. In terms of some biopolymers, the highmolecular weight refers to greater than 100 amino acids or nucleotidesor sugar molecules long.

[0038] The term “bind” includes any physical attachment or closeassociation, which may be permanent or temporary. Generally, aninteraction of hydrogen bonding, hydrophobic interactions, van der Waalsforces, etc., facilitates physical attachment between the molecule ofinterest and the analyte being measured. The “binding” interaction maybe brief as in the situation where binding causes a chemical reaction tooccur. This is typical when the binding component is an enzyme and theanalyte is a substrate for the enzyme. Reactions resulting from contactbetween the binding agent and the analyte are also within the definitionof binding for the purposes of the invention.

[0039] The term “fiber” refers to a filament. A filament or rod may be asolid strand of monolithic, porous, or composite forms, or aggregateforms. Pluralities, typically a large number, of fibers are boundadjacent to each other in ribbons or bundles to form a “fiber bundle”.The cross-section of the fibers may be of any shape, such as round,triangular, square, rectangular or polygonal. A bundle may be sectionedto produce a section piece containing a plurality of thin elements. Thesection piece may be bonded to a substrate to form an array (or asectioned array or microarray). The term array is used here to denoteboth an array and a microarray, as they differ only in size. The termsectioned array is also used to denote how the array is formed by theuse of a sectioned piece of a fiber bundle.

[0040] The term “particle” includes a large number of insolublematerials of any configuration, including spherical, thread-like,brush-like and many irregular shapes. Particles may be porous withregular or random channels inside them. Examples include silica,cellulose, Sepharose beads, polystyrene (solid, porous and derivitized)beads, controlled-pore glass, gel beads, sols, biological cells,viruses, subcellular particles, etc. Even certain high molecular weightmaterials, such as polymers and complexes, may serve as immobilizingstructures that would constitute a “particle”.

[0041] According to the invention, a plurality of fibers may beproduced, with each containing a different agent of interest. The fibermay contain suspended binding agents or it may be coated or impregnatedwith the binding agents. A cut end of the fibers may be briefly treatedwith dilute solvents to expose active groups. In addition, a fiber mayincorporate binding agents according to combinations of the above. Thefibers and binding agents may be used to detect agents of interest in ananalyte, with each fiber being capable of detecting a different agent ofinterest.

[0042] The agent of interest (i.e., a substance to be detected), maycomprise a very broad range of chemicals, complexes, tissues, biologicalcells or fractions thereof. Nucleic acids or proteins, which may havebeen modified or are coated with detergents to make them more soluble inorganic solvents, and a wide range of organic compounds, can beincorporated into mixtures which will solidify, e.g., polymerizingmixtures, such as those used to produce plastics. Oligonucleotides andnucleic acids are soluble in methylene chloride, for example, and hencemay be included in acrylics during polymerization. In the presentinvention, an agent of interest is extracted into an organic solventwhich is miscible with either a thermosetting plastic mixture, or onewhich is polymerized chemically or by UV or ionizing radiation. This maybe done by coating the agents with detergents or other reagents, whichwill make them soluble under the conditions chosen. The solvent may bemiscible in the gelling material or it may be extractable or volatile soas to render a porous final product. Porous products are particularlypreferred with solid filament fibers that are self-supporting.

[0043] Large numbers of different and potentially new active compoundsmay be simultaneously screened by immobilizing them in fibers, bundling,sectioning and forming a microarray. Peak fractions from separations,such as plant extracts, may be simultaneously collected and used to forma microarray. The microarrays may then be used in a large number ofassay systems simultaneously, dramatically reducing the time and effortto screen all of the compounds present for whatever activity onechooses.

[0044] Each fiber used to construct a sectioned array may contain amixture of molecules of interest. For example, during chemicalsynthesis, a number of isomers are prepared. It is convenient to notseparate the isomers before forming a fiber in some circumstances.Likewise, when fractionating a mixture, forming a fiber with a mixtureof receptors may be acceptable, as total and complete isolation isdifficult and time consuming.

[0045] It is equally a part of the present invention to immobilizemicroorganisms or other molecules of interest and use them to localizeantibodies from a patient's sera, and then discover the location of thelatter using a fluorescent anti-human antibody, thus diagnosing adisease which elicited antibody production in the first place.

[0046] When the immobilized macromolecules are antibodies, themicroarray may be used to diagnose a variety of protein-based anomalies.A labeled second antibody to the protein of interest may be used tofurther highlight the cell. In addition, the array may be used toimmobilize infectious agents which have been either previously stainedor which are stained after immobilization. Thus, microbes frombiological samples, e.g. serum or plasma, may be concentrated, stainedwith a fluorescent nucleic acid stain such as TOTO-1 or YOPRO-1, andthen allowed to find their matching antibodies on the array. They maythen be detected by scanning for fluorescence and identified byposition.

[0047] Particularly preferred to be screened are the large numbers ofproteins or peptides generated by mass techniques. Different fractionsfrom a separation technique from a natural source provide a resource ofmany different proteins and peptides. A number of fractionationprocedures are known to separate mixtures of many compounds. Differentfractions or specific compositions may be used to form a single fiber.Two dimensional electrophoresis gels from serum and other tissue andnatural sources produce tens of thousands of different proteinsseparated on the gel. Each may be individually removed (e.g. cut,eluted, etc.) from the gel and used as the molecule of interest to forma single micro-fiber. In such a method, with different bundles beingformed from different samples, protein differences between differentsamples may be readily compared.

[0048] The agent of interest may comprise a very broad range ofchemicals, complexes, biological cells or fractions thereof. Nucleicacids, many proteins, proteins which have been modified or are coatedwith detergents such as sodium dodecyl sulfate are soluble in organicsolvents, and thus can be incorporated into mixtures to be solidified,e.g., polymerizing mixtures such as those used to produce plastics.Hence it is technically feasible to produce long fibers of acrylic orother plastics each containing a different agent of interest usingcurrently available extrusion technology for practice in the presentinvention.

[0049] Arrays have numerous uses other than determining bioactiveproperties. Chemical interactions and reactions may be tested as well,such as an array of different reactive chemicals being tested against atest substance or material to determine corrosion, electrochemicalreaction, or other interaction. This is particularly advantageous in thechemical formulations of plural substances such as in cosmetics, paints,lubricants, etc. Alternatively, one may assay for desirable interactionsbetween the analyte and all of the molecules of interest in the array.

[0050]FIG. 1 shows a tubing 100 containing a fiber 101 formed therein.The fiber 101 may comprise any composition which can solidify whichcontains binding agents. The tubing 100 may be, for example,polyethylene, polypropylene, or fluorocarbon (generic for TEFLON). Thetubing 100 therefore functions as a mold into which a composition whichcan solidify may be inserted and allowed to solidify. In a solidifiedstate, the composition has gelled or hardened, but remains elastic. Inthe past, removing such a fiber 101 from a tubing 100 presented a greatproblem in that the fiber 101 may be somewhat adhered to the insidesurface of the tubing 100 and may be damaged or destroyed duringremoval.

[0051] Examples of compositions which can solidify include, but are notlimited to, polymeric gels, coagulated materials and an IMMUNOBED™(polymethacrylate, polymethylmethacrylate or polyglycolmethacrylate)material, available from Polysciences, Warrington, Pa. A number ofpolymerizing embedding agents have been developed for histological andhistochemical studies, some of which are Durcupan, Nanoplast, Quetol651, London Resin Gold, Lowicryl K4M Polar, Lowicryl Monostep, K4MPolar, Lowicryl K11 Polar, JB-4, JB-4 Plus, IMMUNOBED™, and PolyFreeze,together with data on their composition, curing temperature, solventused and viscosity. Polymers such as agarose, gelatin, collagen,xanthene, carrageenan, alginate, or a thermosetting, thermoplastic,chemosetting or UV polymerizing polymer may be used. Non-polymericgelling materials including waxes and clays may be used. Hydrogels areparticularly preferred when a reaction occurring between the agent ofinterest and an added substance for interrogation requires an aqueousenvironment. These may be further modified by using thickeners, gums,hardening and crosslinking agents, plasticizers and various combinationsof gelling materials. In general, the gelling material should besufficiently inert to not interfere with an interaction between thebinding component and an analyte.

[0052] Immobilization may be accomplished by a number of techniques,known per se, such as entrapment in a matrix or chemically coupling,perhaps through a linking moiety through an amino, hydroxy, sulfhydrylor carboxyl moiety. Chemically attaching the chemical to a monomer to besolidified, e.g., polymerized, also effectively incorporates thecomponent. Binding may also be accomplished by a number of affinitytechniques such as protein A or protein G for antibody attachment,ligand/receptor pairs such as biotin-avidin, HIV-CD4, or through aligand that has a receptor such as digoxigenin-antidigoxigenin.

[0053] The binding between an analyte and the binding agents of thefiber 101 may be enhanced by etching the embedding matrix of each fiber,thereby exposing more of the surface area of particles in each fiber ofthe microarray.

[0054] In addition to methods by which a receptor or molecule ofinterest is immobilized and used to bind an analyte, general methodsexist for arranging immobilized members of a class of reactants. Forexample, protein A or protein G may be immobilized and used tosubsequently bind specific immunoglobulins which in turn will bindspecific analytes. A more general approach is built around the strongand specific reaction between other ligands and receptors such as avidinand biotin. Avidin may be immobilized on a solid support or attached toa gel and used to bind antibodies or other reactants to which biotin hasbeen covalently linked. This allows the production of surfaces to whicha very wide variety of reactants can be readily and quickly attached(see Savage et al., Avidin-Biotin Chemistry: A Handbook, Pierce ChemicalCompany, 1992).

[0055] The immobilized binding components, e.g. nucleic acids, proteins,cells, etc., may be contained in a gel in the tubing 100, may be coatedon the inside of the tubing 100, may be attached to structural elementsembedded within a gel in the tubing 100, or may be coated on orimpregnated in the fiber 101 after it is formed.

[0056] The fibers may contain embedded beads that are capable ofattaching to agents or analytes of interest. The beads may be porous gelbeads used in chromatography such as Sephadex, Biogels and others, orsolid beads such as are used in chromatography. A variety of methods forderivitizing these and for attaching proteins, nucleic acids andpolysaccharides and small molecules thereto have been developed and arewell known to those skilled in the art.

[0057] Different dyes (fluorescent or non-fluorescent) may beincorporated into individual fibers, allowing their location in thetwo-dimensional array to be confirmed.

[0058] The fibers or their gelling material may also contain a dye orother optical absorber so that only analyte/binding components on thesurface of each cell are visualized. Such an improvement reduces theeffects of diffusion rates through a gel or porous material that maychange with temperature, time, type of carrier liquid, etc. A dye thatabsorbs UV or emitted fluorescence will reduce fluorescence fromnon-surface analyte/binding component reactions.

[0059] The embedding matrix for the fibers may be black, opaque orotherwise adsorbent to emitted signals from a label in order to reducecross talk between the cells in the chip. Additionally, any adhesivebetween the fibers may contain the same adsorbent material to reducebackground between cells of the microarray. Optionally, a specific layerof this material may be placed between the fibers before they form thebundle.

[0060] In one embodiment, the composition which can solidify may be areversible polymerizable material that is capable of later beingliquefied. Therefore, a microarray containing such a reversible polymerelement may be used with a test sample. Then the reversible polymerelement may be liquefied and stored or further analyzed. In someapplications this may be more feasible than reading the microarray orremoving the element from the microarray for analysis.

[0061] As an alternative to a homogeneous fiber, a fiber may be preparedwhich incorporates polystyrene latex or other plastic particles (i.e.,beads) onto which proteins or nucleic acids may be attached. Thesupporting plastic may be eroded to a depth of a few microns to revealactive subparticle surfaces, but without dissolving the supportingplastic latex beads. For example, proteins derivatized with fluorinatedgroups attach strongly to fluorocarbon microparticles. Such derivatizedfluorocarbon particles in an acrylic plastic or other suitable embeddingmedium, for example, can be partially exposed at the plastic surface bya dilute acrylic solvent. The solvent may be composed of methylenechloride and ethyl alcohol, for example. The particles or beads may be acomponent of a gelling material, or can be separate components such aslatex beads made of a variety of synthetic plastics (polystyrene, etc.).

[0062] In another alternative non-homogeneous embodiment, thesolidifying, polymerizing or gelling materials may also contain solidstructural elements such as filaments, branched elements, etc., tofurther reinforce or strengthen the gel. Therefore, the fiber 101 mayinclude embedded fibers, strings, threads, particles, objects, etc., toincrease the strength of the fiber 101. The structural elements may alsoprovide attachment sites for the agent of interest. Thus the addedcomponents serve to strengthen the gel, and may provide attachment sitesfor inclusions, including dendrimer branched polynucleic acids, branchedor crosslinked polymeric materials, metal or glass fibers, etc. Threadsor yarn-like and brush-like configurations of structural elements, etc.,may be cast into the length of the fiber giving it strength and allowingthe fiber to be more easily handled or dried. The structural elementsmay serve as the immobilizing component in the fiber for a desiredbinding component. A fiber 101 may therefore be formed with asubstantially central string or thread incorporated therein to increasestrength and to make the fibers 101 easier to handle.

[0063] Binding agents may be attached to the fibers after they areformed. For attachment of the ligands, the rods may be soaked in tubescontaining the substance to be attached or the rods may be coiled upinside a hollow bowl centrifuge rotor having the general configurationof a zonal rotor (see Anderson, N. G., Natl. Cancer Inst. Monograph No.21), but which may be centrifugally drained. The solution of thesubstance to be attached may be centrifuged into the mass and then outof it, followed by washing as necessary. The fibers 101 may then bedried.

[0064] In another alternative embodiment, a fiber 101 may include asecond phase. This second phase may be in the form of, for example,hydrocarbon, aqueous or fluorocarbon microdroplets, particles of sugarsor other water soluble materials, or inorganic particles such as calciumcarbonate particles, which can be dissolved in dilute acids to revealactive groups. Brief exposure of the cut surface of a chip to a solventwill dissolve some of these inclusions, increasing the surface area ofthe support plastic containing the agents of interest.

[0065] The fibers may be identified by tags on the end of the fiber orby tags on the rolls carrying the fibers, and/or by incorporatingdifferent dyes in them. A barcode may also be printed directly near theend of fibers. Thermoplastic polymers may be used when the embeddedproduct is sufficiently thermostable. Some of the fibers may bedifferently colored to assist in the localization of specific ligands inthe array or to identify the array itself.

[0066] A wide variety of methods have been developed to detect reactionsbetween immobilized molecules of interest and soluble reactants. Thesediffer chiefly in the mechanism employed to produce a signal, and in thenumber of different reagents which must be sandwiched together directlyor indirectly to produce that signal. These include fluorescence(including delayed fluorescence) with the fluorescent tag covalentlyattached to the analyte, fluorescence involving soluble dyes which bindto an analyte, and similar dyes whose fluorescence greatly increasesafter binding an analyte. The latter are chiefly used to detect nucleicacids. In more complex systems, including so-called sandwich assays, theend result is the immobilization in the detection complex of an enzymethat, in combination with a soluble substrate, produces a preferablyinsoluble dye that may be fluorescent. Alternatively, the detectioncomplex attached to the bound analyte may include a dendritic molecule,including branching DNA, to which are attached many fluorescent dyemolecules.

[0067] Most immunochemical or competition assays depend on a signalproduced by a reagent other than the analyte. However, methods forfluorescently labeling all proteins containing aliphatic amino groups ina complex mixture have been developed which are reproducible andquantitative. Of these, CyDyes supplied by Amersham Life Sciences, andparticularly, Cy2, Cy3 and Cy5 have proven most useful. When thecomponents of such labeled mixtures are reacted with an array ofimmobilized antibodies, with each being a specific antibody to one ofthe fluorescently labeled analytes, the presence of each of thespecifically bound labeled analytes can be detected by fluorescence.This method can be further improved by exposing the bound antibody arrayto a solution containing known subsaturating quantities of each proteinin a non-fluorescent form, washing the bound antibody array, andexposing it to a test mixture of labeled proteins, thus producing amultiple competition assay.

[0068] Not only can the chips of the present invention be used toidentify infectious agents by identifying characteristic nucleic acidsequences, they can also be used for identifying intact bacteria,mycoplasmas, yeast, nanobacteria, and viruses using arrays ofimmobilized specific antibodies.

[0069] This system may be used for the identification of viruses orother infectious particles isolated by microbanding tubes, as describedin WO99/46047. Thus, microbes from biological samples, e.g. serum orplasma, may be concentrated, stained with a fluorescent nucleic acidstain such as TOTO-1 or YOPRO-1, and then allowed to find their matchingantibodies on the array. They may then be detected by scanning forfluorescence and identified by position. It is equally a part of thepresent invention to immobilize microorganisms or other molecules ofinterest in the described chips, to use them to localize antibodies froma patient's serum, and to then discover the location of the latter usinga fluorescent anti-human antibody, thus diagnosing the disease whichelicited antibody production.

[0070]FIG. 2 shows a cut-away portion 203 of the tubing 100 that hasbeen cut away to expose the end of the fiber 101. The end of the fiber101 may therefore be grasped in some manner.

[0071]FIG. 3 shows the fiber removal according to the present invention.A tensioning device 308, such as a clamp or jaws, for example, may beused to grasp the end of the fiber 101 and place a predeterminedelongation force onto it. The predetermined elongation force may besupplied by hand or by an appropriate mechanical device. Thepredetermined elongation force causes the fiber 101 to elongate andtherefore reduce in diameter, leaving a separation region 305 around theelongated fiber portion. By maintaining the predetermined elongationforce on the fiber 101, the elongation and separation may be propagatedthrough the tubing 100, causing the fiber 101 to eventually breakcompletely free of the tubing 100. However, the predetermined elongationforce must be substantially maintained until the elongation andseparation propagates the full length of the tubing, at which time thefiber 101 may be removed from the tubing 100.

[0072] In a first step of a first method embodiment of forming a fiberaccording to the invention, the composition which can solidify issupplied into the tubing 100. This may be done, for example, byinjecting with a plunger apparatus or by placing a vacuum or partialvacuum on one end of the tubing 100 and placing the other end incommunication with the composition in order to draw it into the tubing100. The tubing 100 may have any desired inner diameter.

[0073] In a second step, the composition is allowed to solidify insidethe tubing 100 in order to form the fiber 101.

[0074] In a third step, a predetermined elongation force is placed onthe fiber 101 in order to cause the elongation and reduction in diameterthat separates the fiber 101 from the tubing 100. The predeterminedelongation force may be supplied by hand or supplied by an appropriatemechanical device. The method may optionally include a step of slittingthe tubing 100.

[0075] In a fourth step, the predetermined elongation force issubstantially maintained until the elongation and separation ispropagated throughout the tubing 100. Fibers thus formed may be used tocreate sectioned arrays of an advantageous size and with a simple andeconomical method of manufacture.

[0076] In a first step of a second method embodiment of forming a fiberaccording to the invention, the composition to be solidified is suppliedinto the tubing 100.

[0077] In a second step, the composition is allowed to solidify insidethe tubing 100 to form the fiber 101.

[0078] In a third step, a portion of the tubing 100 is cut away toexpose an end of the fiber 101. This is done to allow the end to begrasped in some manner.

[0079] In a fourth step, a predetermined elongation force is placed onthe fiber in order to cause the fiber 101 to elongate and separate fromthe inner surface of the tubing 100. The predetermined elongation forcemay be supplied by hand or supplied by an appropriate mechanical device.

[0080] In an optional fifth step, the tubing 100 is slit in theseparation region 305 (the region where the fiber 101 has separated fromthe tubing 100). This may be done in order to more easily remove thetubing 100 from around the fiber 101.

[0081] In a sixth step, the predetermined elongation force is maintainedon the fiber 101 in order to propagate the elongation and separationthroughout the entire length of the tubing 100. This, of course, may bedone by a machine or apparatus that continuously places a force on thefiber 101 to perform a continuous propagation of the elongation andseparation of the fiber 101. In addition, the machine or device maycontinuously slit the tubing 100 in a region following the elongationand separation in order to remove the tubing 100. The tubing 100 may beslit in one or more circumferential positions on the tubing 100,preferably in two substantially opposite circumferential positions.

[0082] In a first step of a third method embodiment of forming a fiberaccording to the invention, the composition to be solidified is suppliedinto the tubing 100 using a plunger apparatus 400 (see FIG. 4). Thecomposition may be supplied into the tubing 100 by injecting or bysucking or aspirating the composition into the tubing 100. The plungerapparatus 400 may be, for example, a syringe that is inserted into oneend of the tubing 100 and that forces the composition into the tubing100 or draws it in by aspiration, in which case the distal end of thetubing 100 is inserted into the composition. The plunger apparatus 400may be suitable for forming non-continuous fibers 101.

[0083] In a second step, the composition is allowed to solidify insidethe tubing 100 to form the fiber 101.

[0084] In a third step, the predetermined elongation force is placed onthe fiber 101 using the plunger apparatus 400. This assumes that theplunger apparatus 400 is placed in and maintained in communication withthe end of the tubing 100 while the composition solidifies. Therefore,the fiber 101 is formed not only in the tubing 100 but continuously intothe plunger apparatus 400. The plunger apparatus 400 may therefore begrasped and used to place the predetermined elongation force on thefiber 101 (making it unnecessary to cut away a portion of the tubing 100for the purpose of grasping the fiber 101). The method may optionallyinclude a step of slitting the tubing 100.

[0085] In a fourth step, the predetermined elongation force ismaintained in order to propagate the elongation and separation of thefiber 101 throughout the tubing 100.

[0086] In any of the method embodiments according to the invention, thefibers thus formed may be used to create one or more sectioned arrays. Aplurality of fibers may be substantially aligned into a bundle and thenthe fibers may be affixed. The present invention makes microarrays or“chips” by sectioning assembled bundles of fibers containing immobilizedbinding components. The fibers may include, for example, biologicalmolecules and entities such as nucleic acid fragments, antigensantibodies, proteins, peptides, carbohydrates, ligands, receptors, drugtargets, biological cells or their subfractions, infectious agents orsubfractions of them, drugs, toxic agents, or natural products. Inaddition, the fibers according to the invention may be composed of twodifferent types of material in coaxial formation.

[0087] There are two basic options for making two-dimensional arraysfrom these fibers. The first is to make and evaluate ribbons, and thento form a set of ribbons into a long rectangular bar, while the secondis to make the bar at the outset. The former option may be moreadvantageous, since the ribbons can be individually evaluated beforebeing formed into a complete array.

[0088] An advantage of the invention is that very large numbers ofarrays may be cut, and some fraction of them may be retained and usedfor standardization. For example, if a bar 100 cm in length wereconstructed, and if the bar were cut at 100 micron intervals, then10,000 section pieces would be available. If the section pieces are 10microns in thickness, then the number of section pieces would be100,000.

[0089] If the individual fibers are 100 microns in diameter, and ifthere are 100 fibers per ribbon, there will be 10,000 fibers in a barhaving a cross-sectional area of 1 cm square. If there are 330 fibersper ribbon, then the total number of fibers in a bar is 108,900,approximately the number of expressed genes postulated to be present inthe human genome.

[0090] The present invention is the first array to have such a largenumber of different cells per unit area on a microarray without thebinding agent being covalently attached to the chip. It is preferred forthe present invention to have at least 100, more preferably 250, 500,1,000, 5,000, 10,000, 100,000 or a million or more cells per squarecentimeter of array. These are much higher concentrations thandepositable cells formed by microfluidics in commercial microarrays.

[0091] To greatly increase the number of cells per square centimeterbeyond even these high numbers, one may prepare a large fiber bundlewith relatively large fibers and stretch or draw the bundle. While thismakes the individual fibers thinner, it does not affect their basiccomposition or their orientation with respect to each other andcross-section geometry. This technique has the twin advantages ofallowing one to make more microarrays and making them smaller. By usingconventional 5 micron porous particles and a plastic embedding mediumsuch as a low melting point wax, the result are deformable or ductilefibers which may be drawn to very thin fibers of less than 20 microns indiameter. The field of drawing thermoplastic materials is well known perse. Even if not truly drawable through a die, one can pull or extrudeplastic materials between rollers to lengthen and reduce the diameter ofthe fibers. With the optional application of gentle heat, one need onlypull the ends of the fiber bundle to generate the same lengthening andreducing of cross-sectional area. With smaller, porous particles, thefibers may be drawn to even thinner dimensions, thereby permittingmicroarrays of up to at least about 10 billion cells per squarecentimeter of microarray.

[0092] The bundle may be affixed in a number of ways. In a first method,the fibers are affixed by casting or embedding the bundle in ahardenable material. A variety of histological embedding media have beendeveloped which preserve nucleic acids and antigens in a reactive form.These include, among others, Durcupan, Nanoplast, Quetrol 651 which maybe cured by very mild heating, JB-4, IMMUNOBED™ which may be polymerizedat room temperature, and the water soluble acrylic polymers London ResinGold and Lowicryl which are polymerized at below freezing temperaturesby ultraviolet light (all are available from Polysciences, Inc.).Conventional embedding media use solvents and waxes, and the waxes mustbe at least partially removed before analysis.

[0093] The embedding material or adhesive used to hold the tubes in abundled configuration may be opaque, while the tubes and preferablytheir contents will conduct light along their length. As a final checkon the orientation of array elements, one element at a time at one endof the bundle may be illuminated, and the light detected and related toarray position at the other end.

[0094] In a second method of affixing the fibers of a bundle, the fibersare affixed by bonding the fibers with an adhesive. A number ofadhesives are known, including cyanoacrylate adhesives. The spacebetween the fibers may be completely filled by adhesive or a monomerwhich is polymerized. Thermoplastic and gelling materials may alsoconstitute the adhesive by causing a large number of fibers to be heldtogether in a block.

[0095] In a third method of affixing the fibers of a bundle, the fibersare affixed by heating the bundle until the fibers soften and bond.Arrays of parallel fibers may be bonded together by many techniques,such as by the introduction of a heated solvent vapor. The vapor isallowed to interact with the array for a specified period of time, andis then removed by re-evacuation. Alternatively, in heat sintering, thebundle of fibers is placed under lateral compression and heated to thesoftening point of the fiber material. Bonding may further beaccomplished by the use of low melting point metals, such as gallium, inan embedding matrix. The matrix may be heated and cooled in order toembed the fibers. By low melting point is meant temperatures at or aboutphysiologic temperature of the binding component. Alternatively, thefibers may be bonded through non-chemical means, such as by passing anelectrical current through the fibers to fuse them.

[0096] In a fourth method of affixing the fibers of a bundle, the bundlemay be encased. This may include wrapping the bundle in a wrapper orplacing the plurality of fibers in a tubing.

[0097] After the fibers are affixed, the bundle may be sectioned. Thebundle may be cut transversely or at an angle into many thin disks andportions are optionally dissolved if desired. Microtomes and othersectioning or cutting instruments capable of cutting assembled bundlesof tubes into thin sections, and of maintaining their orientation aftersectioning, are known. In general, blade cutting is preferred to sawingto reduce contamination of binding components between cells of themicroarray. Microtomes for sectioning soft tissues in wax arecommercially available, as are a variety of techniques and arrangementsfor attaching sections to glass or plastic slides, for treating themautomatically to remove some or all of the embedding media, and forsystematically exposing the slides to a series of reagents.

[0098] A section may be used to complete a sectioned array by bonding oraffixing a section to a substrate. The sections (as microarray chips)may be attached directly to adhesive surfaces on flexible films or onsolid surfaces, such as glass slides. It is also feasible to attachsections (the word “section” is used here in place of “chip”) atintervals along a film strip, with others interleaved between them. Thusa set of about a dozen or more different sections may be placed inrepeating order along the film, and the film then cut up to give oneset. For sequencing studies, one DNA insert sample may be amplified,labeled, and its hybridization to a large set of sections examined.

[0099] With the present invention, a very large number of sections canbe made from one composite assembly, and adjacent sections intercomparedas well as those some distance apart. Statistical analyses will be ableto predict the rate of errors that may occur. However, of even greaterimportance is the fact that since the sections can be made in largenumbers and quite cheaply, it will be feasible to run duplicate analysison clinical samples, and to run confirmatory analysis when importantdiagnostic results are obtained. The present invention therefore makesfeasible widespread and routine application of genetic analyses in thepractice of medicine.

[0100] Essentially the same fiber may be used multiple times in the samemicroarray. This provides an internal quality control check and improvesconfidence in the binding assay. This also provides additionalquantitative measurements if such an assay is performed to improveprecision. Blank fibers with no molecule of interest bound thereto maybe used to provide a good negative control, and should be used in everymicroarray.

[0101] Arrays may have two or more identical cells made from differentfibers but containing identical binding agents. This provides aninternal quality assurance check for the array. Additionally, it ispreferred for some of the cells to provide different concentrations ofthe binding component for quantitative measurement of an analyte. Theseprovide internal standards for the microarray for both qualitativedetection and quantitative detection. For example, a series of cells maycontain different concentrations of an antibiotic in their gels. When asample microorganism is contacted with the cells and allowed toincubate, the absence of growth in one cell and the presence of growthin another cell provide an approximate minimal inhibitory concentration.The same can be done for determining minimal bacteriocidalconcentrations when stained with a vital dye such as trypan blue orfluorescein acetate. Since a microarray may contain thousands of cells,one can simultaneously determine the antibiotic sensitivity to numerousantibiotics simultaneously. Quantitative determination of otherbiological activities with either ligand or receptor immobilized in thegel may be used.

[0102] In the course of using a chip of the instant invention, variousknown techniques and materials are used to reduce non-specific reaction.Thus, in the case of a protein-based assay, the non-specific sites onthe chip contributed by the substance of the fiber or filament, theembedding material, and essentially everything aside from the bindingcomponent of interest are reacted with a blocking agent, such as albuminor milk, so that the blocking agent will bind to those areas notcontaining the binding component which could react with a ligand,analyte, reporter molecule or whatever would specifically bind to thebinding component, as known in the art.

[0103] Since channels are reproducible between plates, the location ofeach channel or cell may be accurately determined by mechanical means.Reference markings on polished edges or other suitable locations mayfurther identify each cell in the array. Current commercially availablecomputer driven two-dimensional drives of sufficient accuracy arecommercially available so that each cell may individually be visualizedor tested, or material may be added thereto or withdrawn therefrom.

[0104] By using the present invention, one avoids the difficulties ofindividually depositing a different reagent on each cell on a solidphase or synthesizing a different compound at each cell. The formertechnique is limited by both the possibilities of spilling and mixingreagents, and by limitations in the accuracy of measurement of smallfluid volumes. The latter technique is limited by the types of differentcompounds that can be synthesized on a solid phase surface. Both priorart techniques are expensive and require elaborate automated equipmentor tedious labor to produce each array individually. By contrast, thepresent invention for producing microarrays is technically simple andquick, and the batch size may be in the thousands. The only individualeffort required for each microarray is the step of cutting.

[0105]FIG. 4 shows a plunger apparatus 400 such as a commonly availablesyringe 400. The syringe 400 includes a chamber 402 and a plunger 404.The syringe may optionally include a needle (not shown) that may becapable of fitting closely into the tubing 100. The chamber 402 may befilled with the composition to be solidified and the plunger apparatus400 may be placed in communication with the tubing 100. The plunger 404may be depressed, forcing the composition into the tubing 100.Alternatively, the opposite end of the tubing 100 can be inserted into asolution of the composition, which can be drawn into the tubing 100 andchamber 402 by aspiration caused by withdrawing the plunger 404. Theplunger apparatus 400 may be left in communication with the tubing 100until the material has solidified.

[0106] In one embodiment, the plunger apparatus 400 may comprise a 16gauge needle attached to a 3 cc (cubic centimeter) disposable syringe.The needle may be inserted into a length of polyethylene tubing 100having a 1.5 millimeter internal diameter, for example. A prepolymersolution may be prepared by mixing 25 parts of catalyzed IMMUNOBED™Solution A with 1 part of IMMUNOBED™ Solution B (both available fromPolysciences, Warrington, Pa). The non-polymerized IMMUNOBED™ materialmay then be injected or aspirated into the tubing 100.

[0107]FIG. 5 shows an apparatus 500 for forming an essentiallycontinuous fiber 101. A supply reel 501 includes a length of tubing 100containing a fiber 101 therein. The supply reel 501 feeds into a knifeblock 523, which is used to separate the fiber 101 from the tubing 100and to slit the tubing 100. After passing through the knife block 523,the tubing 100 has been split into pieces 100′ (preferably two pieces)which pass over the tensioning rollers 530 and onto the pick up reels534.

[0108] The tubing 100 and encased fiber 101 may alternatively be fedinto the knife block from a different source, such as, for example, aprocess or machinery for inserting or injecting the composition to besolidified into the tubing 100.

[0109] Before reaching the knife block 523, the tubing 100 passes over afirst feed device 503 that includes a first roller 508 and a pair ofsecond rollers 511. The first feed device 503 may be adjusted and movedin order to feed the tubing from the supply reel 501. The movement isperformed by a screw device 515 which may include a motor 516 that canposition the first feed device 503 (vertically, in the embodimentshown). A first pair of stationary rollers 519 maintains a constant feedheight of the tubing into the knife block 523. The tubing passes througha hole in the knife block 523. The tubing 100 and encased fiber 101 arepulled through the knife block 523 by a tension applied to the finishedfiber 101′ and a tension applied to the split tubing pieces 100′. Thepulling in a preferred embodiment is achieved mainly through tensionapplied to the finished fiber 101′, although various tensions may beapplied to either the finished fiber 101′ or the split tubing pieces100′.

[0110] After splitting in the knife block 523, the finished fiber 101′passes through a second pair of stationary rollers 537 and into a secondfeed device 539, which feeds the finished fiber 101′ onto the take-upreel 560.

[0111] The second feed device 539, like the first feed device 503,includes a first set of rollers 542 and a second roller 546. The secondfeed device 539 is moved by a screw device 551 and motor 552 that may beused to feed the finished fiber 101′ onto the take-up reel 560(vertically, in the embodiment shown).

[0112] The rollers 537 and 542 may be formed of fluorocarbon or may befluorocarbon coated in order to prevent the finished fiber 101′ fromadhering to the rollers.

[0113] The knife block 523 may be comprised of two joined, symmetricknife block halves 523′. The two knife block halves 523′ may be joinedby fasteners 526 (such as by screws, bolts, etc.) and the adjustmentknob or crank 527 may be used to control the slitting operation insidethe assembled knife block 523, as will be explained below in conjunctionwith FIGS. 6-8.

[0114] The separation process may be started by hand, such as by cuttingthe tubing 100 and exposing an end of the fiber 101 before feeding itinto the apparatus 500. Alternatively, the plunger apparatus 400 may beused to start the separation process. However, it should be understoodthat other starting procedures may be employed in order to initiate theseparation process performed using the apparatus 500.

[0115] The apparatus 500 may be under the control of a computer (notshown) in order to control the feeding, splitting, and take-upoperations. The computer may additionally perform tensioning of thevarious reels and rollers.

[0116] It should be understood that the finished fiber 101′ produced bythe methods and apparatus according to the invention may be sectionedand formed into sectioned arrays or microarrays. These sectioned arraysmay be advantageously smaller and denser than a sectioned array formedaccording to the prior art (sectioning and using a fiber 101 stillinside the tubing 100).

[0117]FIG. 6 shows detail of one knife block half 523′. The knife blockhalf 523′ includes a groove 604 of a predetermined size approximatelyequal to the outside diameter of the tubing 100. The groove 604 mayinclude bevels 605 at the ends to aid in the entry and exit of thetubing 100. The tubing 100 is, therefore, fed through the hole made bythe grooves 604 in the two knife block halves 523′.

[0118] The knife block half 523′ also includes one or more milledcutouts 608. The one or more cutouts 608 accommodate one or more knifeblades 714 used for the slitting operation, as will be shown in FIG. 7.The knife block half 523′ also includes one or more recesses 612 andholes 616 for one or more cut-depth adjusters 527 (discussed inconjunction with FIG. 8). Holes 620 accommodate fasteners, such as boltsor screws, that are used to join the two knife block halves 523′.

[0119]FIG. 7 shows detail of one knife block half 523′ with one or moreknives 714 in place. A knife 714 may be, for example, a single-edgerazor blade. The at least one knife 714 has an edge 715 that may bepositioned so that it protrudes into the groove 604. The amount ofprotrusion into the groove 604 will determine the depth of the cut intothe tubing 100. It should be noted that the at least one knife 714should preferably not cut into the fiber 101, but should only cutthrough the tubing 100. The at least one knife 714 , therefore, may bepositioned so that when the at least one cut depth adjuster 527 is inplace in the at least one cut depth adjuster recess 612, the at leastone cut depth adjuster 527 can control how far into the groove 604 theat least one knife 714 protrudes, and can therefore control the cutdepth.

[0120]FIG. 8 shows the cut depth adjuster 527. As illustrated in FIG. 5,the crank or knob portion 819 and the plate 816 are positioned on theoutside of the assembled knife block 523. The shaft 805 fits into thehole 616 in the recess 612 of a knife block half 523′, while the offsetlobe 810 rests in the recess 612. When the knob or crank 819 is rotated,the offset lobe 810 displaces the knife 714, thereby adjusting how farthe cutting edge 715 of the knife 714 protrudes into the groove 604.

[0121] The fiber produced according to the various methods and apparatusof the invention may then be grouped into a bundle and sectioned or cutinto thin slices. The bundle may be substantially rectangular incross-section, may be substantially circular, substantially ovoid, oreven irregular. The slices may be bonded to substrates in order to formsectioned arrays or microarrays. The substrate may be a flexible film ora solid surface, such as glass, for example. The substrate may betransparent so that the sectioned array may be illuminated by light.

[0122] Various methods of array manufacturing currently exist. In onemethod, a plurality of fibers are created. The fibers are impregnated orcoated with a variety of binding agents that bind to the molecules ormacromolecules sought to be detected. The resulting fibers are groupedinto a bundle and fastened together. Very thin slices of the bundle aresectioned (cut off) and glued or bonded to a substrate in order to forman array. The array and substrate are commonly referred to as a chip.The completed chip may be exposed to a test sample and then read, suchas by an apparatus having a light source and a light detector that readsthe pattern of light emitted or transmitted from the chip. The resultinglight pattern will be modulated by the molecules or macromolecules thathave bonded to the binding agents on the chip. The resulting lightpattern will indicate the presence or absence of certain molecules ormacromolecules.

[0123] For general clinical use it is important to have identifiers onthe slide holding the chip, and identifiers may be integral with thechip itself. This may include a barcode printed along an edge or borderof a chip in order to provide identification and orientation. Inaddition, small concentrations of dyes, usually non-fluorescent, may beincorporated into selected fibers to identify them or to present apattern. It may also be useful to incorporate fluorescent dyes intoselected cells or elements, and which serve to calibrate thefluorescence measurements.

[0124] Dyed fibers would be visible in such arrays to confirmidentifications and orientation. In addition, the fibers can be dyed insuch a manner that a visible pattern is formed if the array is correctlymade, and the pattern may include a name or a number.

[0125] Additional fibers or ribbons may be added to the bundle as neededbefore sectioning additional arrays. This allows one to detect andmeasure newly discovered emerging diseases, new proteins, genes orcompounds without recreating a completely new bundle.

[0126] This invention may be applied in an alternative fashion in whichthe bundles are stored at user sites, and the arrays only sliced off asneeded. This arrangement may be useful for research purposes whereidentical arrays are required over the long term, but only a few arerequired at any one time.

[0127] Another alternative to slicing the bundle and using its sectionsas separate microarrays is to perform the assay with the end of thebundle directly. After the assay is performed wherein a first samplecould be applied to the cut cross-sectional surface, and washed off, adetector could image the result. One may then mount the bundle in amicrotome device, if the bundle were not already so mounted before theassay. A blade could then remove the used surface of the bundle,exposing a fresh surface for the next assay, which would repeat the samesteps again. The bundle could thus be used in one machine for a seriesof up to 100,000 or more assays performed one after another. Thisarrangement has certain advantages as optical or electrical detectionmay be performed through the bundle itself with fiber optic fibers orconductive fibers. The detection system may be continuously attached tothe bundle while a more general light or electrical energy is applied tothe end being used for testing.

[0128] The invention allows different immobilization technologies,different classes of immobilized agents of interest, different classesof analytes, and different types of detection methodologies to beemployed on a single chip.

[0129] In a first example, a fiber is produced according to theinvention with the fiber containing non-covalently entrapped biologicaltarget molecules. In this example, an element of a microarray is formedby mixing a biological target molecule with a substance that can besubsequently removed from the element of the microarray, allowing thebiological target molecule to then react with a component of a surfacewith which it is in contact, to provide a stable linkage between thebiological target molecule and the surface. More specifically, a proteincan be used for this purpose that has a recognition site for anothermolecule, such as biotin, which can be bound by streptavidin. Thisbiotinylated-protein can be mixed with a reversible gelling system, suchas, but not limited to, agarose. Fibers can be formed in which theelements are comprised of the reversible gel containing a biologicaltarget molecule. Once formed into an array, thin sections can beprepared and mounted on a surface that contains immobilized recognitionfactors, such as in this case streptavidin. The gel can then bedissolved or removed by any means to expose the biological targetmolecule, which is then free to diffuse and react with the immobilizedrecognition factor. This has the advantage of eliminating the supportpolymer as a barrier to reactants and can serve to increase theprocessing time for analyte detection. The recognition system can becomprised of many types of interactions, such as, but not limited to,antigen-antibody, lectin-carbohydrate, and, in general, any of thewell-known ligand-receptor systems. Reversible gels can be comprised of,but are not limited to, heat-reversible agar or agarose systems,metal-dependent alginate systems, redox-dependent disulfide-containingpolymeric systems, e.g., polymers formed by oxidation of sulfhydrylgroups to disulfides that can be reduced back to free sulfhydryl groups.In addition, the support matrix may be one that can be degraded by anymeans to liberate the entrapped biological target molecule. Thedegradation process can consist of, but would not be limited to acid orbase hydrolysis, enzymatic hydrolysis, photo degradation, temperaturechange such as with thermal responsive polymers which are solid orliquid, depending on the temperature, and other processors known in theart.

[0130] In a second example, a fiber is produced according to theinvention with the fiber containing biological target moleculesincorporated by diffusion after polymerization of the fibers. In thisexample, an element of a microarray is formed by incorporating abiological target molecule into the fiber by diffusion from a solutioncontaining the biological target molecule. This example provides a meansof incorporating labile biological target molecules into polymericmatrices in those cases where the conditions of polymerization, such asheat, presence of free radicals, etc., are capable of inactivating thebiological target molecule. A fiber is prepared from a material such as,but not limited to, IMMUNOBED™. The fiber is placed in a solutioncontaining a biological target molecule of interest. The fiber isallowed to remain in contact with the solution for a period of time, ata temperature that is consistent with maintenance of biologicalactivity, generally from, but not restricted to, 4° C. to 40° C. Theperiod of time of contact between the fiber and the solution for optimalincorporation of the biological target molecule will depend on manyfactors, including porosity of the fiber, molecular size of thebiological target molecule, concentration of the biological targetmolecule, temperature, etc. After the fiber is loaded with biologicaltarget molecule it is washed with a buffer and incorporated into anarray, as previously described.

[0131] In a third example, a fiber is produced according to theinvention with the fiber containing biological target moleculesincorporated by diffusion and entrapment after polymerization of thefibers. In this example, a biological target molecule is incorporatedinto a fiber by diffusion from a solution containing the biologicaltarget molecule. This example provides a means of incorporating labilebiological target molecules into polymeric matrices in those cases wherethe conditions of polymerization, such as heat, presence of freeradicals, etc., are capable of inactivating the biological targetmolecule. It also provides a means for entrapping the biological targetmolecule within the polymeric fiber to prevent the subsequent diffusionof the biological target molecule out of the fiber. A polymeric fiber ofmaterial is prepared from a material such as, but not limited to,IMMUNOBED™. The mixture of monomeric substances is mixed with anentrapping agent, such as, but not limited to, bovine serumalbumin-biotin complex. The mixture is formed into a fiber, and thefiber is placed in a solution containing a biological target molecule ofinterest. In this case the biological target molecule of interest willbe conjugated to a biotin-binding protein such as streptavidin. Otherbinding pairs, which are known in the art, can also be used for thispurpose. The fiber is allowed to remain in contact with the solution fora period of time, at a temperature that is consistent with maintenanceof biological activity, generally from, but not restricted to, 4° C. to40° C. The period of time of contact between the fiber and the solutionfor optimal incorporation of the biological target molecule into thefiber will depend on many factors, including porosity of the fiber,molecular size of the biological target molecule, concentration of thebiological target molecule, temperature, etc. After the fiber is loadedwith biological target molecule it is washed with a buffer andincorporated into an array, as previously described.

[0132] While the invention has been described in detail above, theinvention is not intended to be limited to the specific embodiments asdescribed. It is evident that those skilled in the art may now makenumerous uses and modifications of and departures from the specificembodiments described herein without departing from the inventiveconcepts.

What is claimed is:
 1. A method for removing an elastic temporarilydeformable fiber from a mold, comprising the steps of: supplying acomposition into a mold; allowing said composition to solidify in saidmold and form said fiber; placing a predetermined elongation force ontoan end of said fiber, said predetermined elongation force causing anelongation and reduction in cross-section of said fiber and causing aseparation of said fiber from an interior surface of said mold; andsubstantially maintaining said predetermined elongation force topropagate said separation through said mold until said fiber iscompletely separated from said interior surface of said mold.
 2. Themethod of claim 1 wherein said composition is a polymerizablecomposition.
 3. The method of claim 1, wherein said supplying andplacing steps are performed by a plunger apparatus communicating withsaid mold.
 4. The method of claim 1, wherein said composition includes abinding agent.
 5. The method of claim 1, wherein said compositionincludes non-polymer reinforcing elements.
 6. The method of claim 1,wherein said composition is a reversible polymerizable composition. 7.The method of claim 1 wherein said mold is tubing.
 8. The method ofclaim 7, wherein said mold comprises fluorocarbon tubing.
 9. The methodof claim 7, wherein said mold comprises polyethylene tubing.
 10. Themethod of claim 7, wherein said tubing comprises polypropylene tubing.11. The method of claim 1, wherein said composition comprises a gellingmaterial.
 12. The method of claim 1, wherein said composition comprisesa coagulating material.
 13. The method of claim 1, wherein saidcomposition comprises polymethacrylate, polymethylmethacrylate orpolyglycolmethacrylate.
 14. The method of claim 1, further comprisingthe step of slitting said mold.
 15. The method of claim 1, furthercomprising the step of slitting said mold in at least two substantiallyopposite circumferential positions.
 16. The method of claim 1, furthercomprising the step of cutting said end of said fiber to expose saidend, said cutting occurring prior to placing said predetermined forceonto said fiber.
 17. The method of claim 1, wherein said composition issupplied into said mold by injecting said composition into said mold.18. The method of claim 1, wherein said composition is supplied intosaid tubing by aspirating said composition into said tubing under avacuum.
 19. The method of claim 16 wherein said aspirating occurs bycapillary action.
 20. A method for making a fiber, comprising the stepsof: supplying a composition into a mold; allowing said composition tosolidify in said mold and form said fiber; exposing an end of saidfiber; placing a predetermined elongation force onto said end of saidfiber, said predetermined elongation force causing an elongation andreduction in cross-section of said fiber and causing a separation ofsaid fiber from an interior surface of said mold; slitting said mold;and substantially maintaining said predetermined elongation force topropagate said separation through said mold until said fiber iscompletely separated from said interior surface of said mold.
 21. Themethod of claim 20 wherein said composition is a polymerizablecomposition.
 22. The method of claim 20, wherein said compositionincludes a binding agent.
 23. The method of claim 20, wherein saidcomposition includes non-polymer reinforcing elements.
 24. The method ofclaim 20, wherein said composition is a reversible polymerizablecomposition.
 25. The method of claim 20, wherein said mold is tubing.26. The method of claim 25, wherein said tubing comprises fluorocarbontubing.
 27. The method of claim 25, wherein said tubing comprisespolyethylene tubing.
 28. The method of claim 25, wherein said tubingcomprises polypropylene tubing.
 29. The method of claim 20, wherein saidcomposition comprises a gelling material.
 30. The method of claim 20,wherein said composition comprises a coagulating material.
 31. Themethod of claim 20, wherein said composition comprises polymethacrylate,polymethylmethacrylate or polyglycolmethacrylate.
 32. The method ofclaim 20, wherein two substantially opposite sides of said mold areslit.
 33. The method of claim 20, wherein said slitting is performed byfeeding said mold and said fiber through a knife block containing atleast one knife.
 34. The method of claim 20, wherein said slitting isperformed in a region of said mold where said fiber has separated fromsaid mold.
 35. The method of claim 20, wherein said composition issupplied into said mold by injecting said composition into said mold.36. The method of claim 20, wherein said composition is supplied intosaid mold by aspirating said composition into said mold under a vacuum.37. The method of claim 36 wherein said aspirating is by capillaryaction.
 38. A method for making a fiber, comprising the steps of:supplying a composition into a mold using a plunger apparatuscommunicating with said mold; allowing said composition to solidify andform said fiber in said mold and to solidify in said plunger apparatus;placing a predetermined elongation force onto an end of said fiber usingsaid plunger apparatus, said predetermined elongation force causing anelongation and reduction in cross-section of said fiber and causing aseparation of said fiber from an interior surface of said mold; andsubstantially maintaining said predetermined elongation force topropagate said separation through said mold until said fiber iscompletely separated from said interior surface of said mold.
 39. Themethod of claim 38 wherein said composition is a polymerizablecomposition.
 40. The method of claim 38, wherein said compositionincludes a binding agent.
 41. The method of claim 38, wherein saidcomposition includes non-polymer reinforcing elements.
 42. The method ofclaim 38, wherein said composition is a reversible polymerizablecomposition.
 43. The method of claim 38 wherein said mold is tubing. 44.The method of claim 43, wherein said tubing comprises fluorocarbontubing.
 45. The method of claim 43, wherein said tubing comprisespolyethylene tubing.
 46. The method of claim 43, wherein said tubingcomprises polypropylene tubing.
 47. The method of claim 38, wherein saidcomposition comprises a gelling material.
 48. The method of claim 38,wherein said composition comprises a coagulating material.
 49. Themethod of claim 38, wherein said composition comprises polymethacrylate,polymethylmethacrylate or polyglycolmethacrylate.
 50. The method ofclaim 38, further comprising the step of slitting said mold.
 51. Themethod of claim 38, further comprising the step of slitting said mold inat least two substantially opposite circumferential positions.
 52. Themethod of claim 38, wherein said composition is supplied into said moldby injecting said composition into said mold.
 53. The method of claim38, wherein said composition is supplied into said mold by aspiratingsaid composition into said mold under a vacuum.
 54. The method of claim53 wherein said aspirating is by capillary action.
 55. A method formaking a fiber and a sectioned array, comprising the steps of: supplyinga composition into a mold; allowing said composition to solidify in saidmold and form said fiber; placing a predetermined elongation force ontoan end of said fiber, said predetermined elongation force causing anelongation and reduction in cross-section of said fiber and causing aseparation of said fiber from an interior surface of said mold;substantially maintaining said predetermined elongation force topropagate said separation through said mold until said fiber iscompletely separated from said interior surface of said mold;substantially aligning a plurality of fibers into a bundle; affixingfibers of said bundle; sectioning said bundle; and affixing a sectionpiece to a substrate to form said sectioned array.
 56. The method ofclaim 55 wherein said composition is a polymerizable composition. 57.The method of claim 55, wherein said supplying and placing steps areperformed by a plunger apparatus communicating with said mold.
 58. Themethod of claim 55, further comprising the step of slitting said mold.59. The method of claim 55, wherein said affixing comprises casting saidbundle in a hardenable material.
 60. The method of claim 55, whereinsaid affixing comprises bonding said fibers with an adhesive.
 61. Themethod of claim 55, wherein said affixing comprises heating said bundleuntil said fibers bond.
 62. The method of claim 55, wherein saidaffixing comprises encasing said bundle.
 63. The method of claim 55,wherein said composition is supplied into said mold by injecting saidcomposition into said mold.
 64. The method of claim 55, wherein saidcomposition is supplied into said mold by aspirating said compositioninto said mold under a vacuum.
 65. The method of claim 64 wherein saidaspiration occurs by capillary action.
 66. A sectioned array produced bythe method of claim
 55. 67. A fiber made by the method of claim
 20. 68.A bundle of fibers comprising fibers of claim
 67. 69. A fiber made bythe method of claim
 38. 70. A bundle of fibers comprising fibers ofclaim
 69. 71. An apparatus for making a fiber, comprising: a knife blockhaving a passage therethrough of a size to accommodate a tubing having afiber therein; at least one knife adjustably held in said knife blockand positionable so that said at least one knife slits said tubing whensaid tubing is fed through said knife block; and a tensioning devicecapable of gripping said fiber and placing a predetermined elongationforce on said fiber; wherein said predetermined elongation forcepropagates an elongation and reduction in cross-section of said fiber insaid tubing and causes a separation of said fiber from an interiorsurface of said tubing, and said predetermined elongation force alsopulls said tubing and said fiber through said knife block, where saidtubing is slit.
 72. The apparatus of claim 71, further comprising one ormore rollers that guide said fiber after said separation.
 73. Theapparatus of claim 71, further comprising one or morefluorocarbon-coated rollers that guide said fiber after said separation.74. The apparatus of claim 71, wherein said tensioning device comprisesa pair of jaws capable of gripping and pulling said fiber.
 75. Theapparatus of claim 71, wherein said tubing and said fiber are fed intosaid knife block from a supply reel.
 76. The apparatus of claim 71,wherein said tensioning device comprises a take-up reel.
 77. Theapparatus of claim 71, wherein said fiber is accumulated on a take-upreel.
 78. The apparatus of claim 71, wherein slit tubing is accumulatedon at least one pick-up reel.
 79. The apparatus of claim 71, whereinsaid knife block further comprises a pair of knife block halves capableof being fastened together, with each knife block half including aportion of said passage and at least one knife cutout to accommodatesaid at least one knife.
 80. The apparatus of claim 71, wherein a cutdepth of said at least one knife is adjustable.
 81. The apparatus ofclaim 71, wherein said knife block includes at least one rotatable cutdepth adjuster including an offset lobe, and said at least one rotatablecut depth adjuster adjusts a cut depth when rotated in contact with saidat least one knife.
 82. The apparatus of claim 71, wherein said at leastone knife comprises a single-edge razor blade.
 83. The apparatus ofclaim 71, wherein said knife block includes two knives positioned toslit substantially opposite sides of said tubing.